Sigma factors are bacterial transcription initiation factors, which bind to the bacterial "core" RNA polymerase and impart on the resulting "holo" RNA polymerase the ability to recognize promoter DNA sequences and orchestrate the strand separation of promoter DNA required for establishment of the initiation-competent "open" RNA polymerase-promoter complex. The focus of this application is on the role of sigma factor in formation of the open complex. With initiation of RNA synthesis being the major point of regulation of gene expression, the biological relevance of our studies stems from the insights into this process that our work will provide. [unreadable] [unreadable] We had previously demonstrated that particular aromatic and basic residues in conserved region 2.3 of the main sigma factor of E. coli, sigma70, were crucial to the DNA strand separation process. We proposed that these residues initiated strand separation by facilitating the rotation of the -11A of the promoter out of the DNA helix and into a binding pocket on RNA polymerase. This model will be further tested by using fluorescence techniques. [unreadable] [unreadable] In studies on sigma32, the heat shock sigma factor of E. coli, and sigmaA, the main sigma factor of the thermophilic bacterium, T. aquaticus, we will explore the generality of the principles established for sigma70, with emphasis on the DNA melting function of region 2.3. In this region, sigmaA is almost identical to sigma70, but sigma32 is different at about half the amino acid residues. The importance of residues in region 2.3 will be assessed by determining the effects of alanine substitutions on open complex formation and on the interaction of RNA polymerase with several model DNAs. We will also develop a single-molecule assay for the interaction of RNA polymerase with promoter DNA, which should allow intermediate promoter complexes to be more readily detected and characterized than possible with current techniques. [unreadable] [unreadable]